An urgent need exists to prevent the sexual transmission of HIV-1 to women. Worldwide, 70% of new cases are spread by sexual intercourse, with women more likely to be infected than men. Using a Multipurpose Prevention Technologies approach, we will test the overall hypothesis that some chemical contraceptives act directly on female reproductive tract (FRT) CD4+T cells and macrophages and indirectly through epithelial cells and fibroblasts in the absence or presence of inflammation to decrease availability of TFV diphosphate (TFV- DP), the biologically active form of TFV and TFV alafenamide (TAF), and thereby increase the risk of HIV infection in women. Since newer progestational chemical contraceptives (LNG: Levonorgestrel and NET: norethisterone) have fewer side effects than medroxyprogesterone acetate (MPA), central to this proposal is the comparison of MPA to LNG and NET, and their effects on both TFV and TAF, the likely next generation microbicide that better targets lymphoid tissues and cells through enhanced uptake and subsequent conversion to TFV. This proposal has 3 Aims that test the following hypotheses: Aim 1. Chemical contraceptives act directly to alter TFV-DP intracellular levels in HIV-target cells in human FRT tissues in ways that compromise microbicide protection against HIV infection. Aim 2. Chemical contraceptives regulate TFV-DP concentrations in HIV-target cells through mechanisms that alter the enzymes necessary for the formation and/or degradation of TFV-DP. Aim 3. Inflammatory factors (PRR Ligands) and chemical contraceptives alter intracellular availability of TFV and TAF and compromise innate immune responses by HIV-target cells from the FRT. This study is unique in that it integrates our understanding of the endocrine and immune systems in the human FRT, as it relates directly to intracellular TFV-DP concentrations in the very cells most likely to be infected by HIV. Our goal is to determine how the next generation of chemical contraceptives (LNG or NET) influence microbicide (TFV and TAF) availability in FRT HIV-target cells and how inflammatory factors compromise these effects. Building on our past research, we expect that these studies will demonstrate that some chemical contraceptive/microbicide combinations, will compromise microbicide efficacy and protection against HIV infection especially in the presence of inflammatory mediators. These studies will provide a foundation of information essential for the development of the next generation of combined chemical contraceptives and microbicides needed to provide both contraception and improved protection against HIV infection.